ThermoGen proposes to develop a genetic transfer system for gram negative aerobic extreme thermophiles based both on plasmids from these organisms and on chromosomal delivery systems characterized in Phase I. The gene transfer system will not only function as an important genetic tool for the manipulation of these organisms and their uniquely stable enzymes, but will allow the rapid transfer of genes between species spanning vastly different growth conditions. The new system will allow DNA cloning by complementation and gene tagging in the extreme thermophiles. The facilitation of thermophilic gene cloning will enhance the development of thermophilic enzyme reactors with potential applications in therapeutics, food processing, industrial processes, toxic waste control, and general research. The system will also allow selection of thermostable mutations in previously cloned genes. By shuttling the gene of interest from a moderate thermophile to a more extreme thermophile, thermostable activity can be gradually screened or selected for. The new system will aid in the study of thermophily, giving insight to rational redesign of currently used proteins to enhance their function and stability. An available gene transfer system will aid in the discovery of new biochemical pathways with potential commercial and therapeutic applications.